R2A/MAC Biopaddles™ Colony Identification App Code 5540

LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

COMING SOON! R2A/MAC BioPaddles™ Colony Identification App Code 5540

R2A Agar (R2A) MacConkey Agar (MAC)

USE: Enumeration and cultivation of bacteria from potable water; total coliform testing (TCC). Coliform Testing / Recovery of Stressed Coliforms

Side 1: R2A Agar (R2A) (colorless) (Side 1 is marked with an indented laser line)

Side 2: MacConkey Agar (MAC) (pink)

APPLICATION For bacterial plate counts of treated potable water as well as oligotrophic heterotrophs. In total coliform testing (TCC), the coliform organisms tested for include: total coliform, fecal coliform, and E. coli (Escherichia coli). Detection of fecal coliforms (a subset of total coliforms) or Escherichia coli (a subset of fecal coliforms) can indicate the potential presence of waterborne pathogens associated with fecal contamination.1 R2A Agar is recommended in Standard Methods for the Examination of Water and Wastewater for pour plate, spread plate and membrane filter methods for heterotrophic counts.

PADDLE AGARS R2A Agar (R2A) – Developed by Reasoner and Geldreich2 for bacterial plate counts of treated potable water as well as oligotrophic (thrive in low-nutrient conditions) heterotrophs. R2A Agar, in combination with a lower incubation temperature and longer incubation time, stimulates the growth of stressed and chlorine-tolerant bacteria. Enzymatic digest of casein and animal tissue provides nitrogen, carbon and minerals in R2A Agar. Yeast extract is a source of vitamins and trace elements. Dextrose serves as a carbon source. Soluble starch aids in the recovery of injured organisms by absorbing toxic metabolic by-products. Dipotassium Phosphate is used to balance the pH, and magnesium sulfate heptahydrate is a source of divalent cations and sulfate. Sodium pyruvate increases the recovery of stressed cells. Agar and a proprietary polymer are the solidifying agents.

NOTE: Fast-growing bacteria may produce smaller size colonies on R2A Agar than on nutritionally rich media. R2A Agar is a low nutrient medium intended for culturing compromised microorganisms.

1 United States Pharmacopeial Convention. 2007. The United States pharmacopeia, 31st ed., Amended Chapters 61, 62, 111. The United States Pharmacopeial Convention, Rockville, MD. 2 Reasoner, D. J., and E. E. Geldreich. 1979. A new medium for the enumeration and subculture of bacteria from potable water. Abstracts of the Annual Meeting of the American Society for Microbiology 79th Meeting, Paper No. N7.

For in vitro diagnostic use only. This product should be used only by adequately trained personnel with knowledge of microbiological techniques in the laboratory. ©LaMotte BioPaddles. All rights reserved. LaMotte_BioPaddles_R2A_MAC Page | 1 LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

MacConkey Agar (MAC) – Both selective and differential; used to differentiate between Gram negative bacteria while inhibiting the growth of most Gram positive bacteria. The medium also differentiates between lactose-fermenting coliforms Lac (+) and lactose non-fermenters Lac (-), which include potential pathogens. Addition to the nutrient agar base of bile salts and crystal violet will inhibit the growth of most Gram-positive bacteria, making MacConkey agar selective. Lactose, a fermentable carbohydrate, and neutral red, a pH indicator, are added to differentiate the lactose positive coliforms from the potentially pathogenic lactose non-fermenters. When lactose is fermented, acid products lower the pH below 6.8, with the resulting colonial growth turning pinkish-red. If an organism is unable to ferment lactose, the colonies will be colorless. Bile salts mixture and crystal violet are the selective agents, inhibiting Gram positive cocci and allowing Gram-negative organisms to grow. Sodium Chloride maintains the osmotic environment. Agar and a proprietary polymer are the solidifying agents.

CULTURE CONTROLS 10-300 inoculum (CFU) R2A Agar MacConkey Agar Bacillus subtilis GROWTH INHIBITED Enterococcus faecalis GROWTH INHIBITED Escherichia coli GROWTH GROWTH Pseudomonas aeruginosa GROWTH GROWTH Staphylococcus aureus GROWTH GROWTH

STORAGE/EXPIRATION Store tightly sealed BioPaddles™ in a cool, dry location (less than 68°F/20°C). Avoid temperature changes. BioPaddles™ may be refrigerated, but it is not necessary. Do not freeze. If freezing occurs, thaw (3-6 hours) under refrigeration temperatures (40°F; 4.4°C). Freezing can promote excess water loss and variation in media surface due to crystal formation. The average shelf-life is one year. Refer to expiration date (See: BBE stamped on vial). Discard if paddle agar appears oxidized (darkens from expected color). The expiration date applies to the medium in an intact container when stored as directed.

SAMPLING Liquids: Twist to remove paddle from vial. Fill vial to 40 mL fill line with the liquid to be sampled. The 40 mL volume can be used to calculate Total Viable Count (TVC) and/or Total Colony Count (TCC). Replace paddle. Allow a contact time of 15 seconds. Remove the paddle. Empty the vial. Replace the paddle in the vial.

Surfaces: Twist to remove paddle from vial. Allow the paddle surface (10 cm2) to come into physical contact with the test surface. Recovery rate is about 50%. To insure an accurate recovery, gently sweep (or touch) the paddle to cover a 20 cm2 area. Replace paddle in vial.

For in vitro diagnostic use only. This product should be used only by adequately trained personnel with knowledge of microbiological techniques in the laboratory. ©LaMotte BioPaddles. All rights reserved. LaMotte_BioPaddles_R2A_MAC Page | 2 LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

INCUBATION Incubate at 37°C for 24 hours, continue at 25°C for 3-5 days.

NOTE: Fast-growing bacteria may produce smaller size colonies on R2A Agar than on nutritionally rich media. R2A Agar is a low nutrient medium intended for culturing compromised microorganisms.

COLONY MEASURING Each BioPaddles™ paddle has molded media attachment points that are 4mm in length (point-to-point). This feature provides a useful guidepost to estimating nearby colony size.

IDENTIFICATION ORGANISM R2A MAC

ORGANISM PHYSIOLOGY GROWTH COLONY IMAGE GROWTH COLONY IMAGE ◆ Precision Test Strip Available Aspergillus niger • Catalase (+) • Granular • Ascomycete • Jet black conidia w/ INHIBITED ------+++ yellow/gray hyphae • 3-5++cm Bacillus spp. • Lactose (-) • Translucent to • Indole (-) ◆ dull, off-white; • Oxidase (-) ◆ opaque • PARTIAL - • Catalase (+) ◆ Smooth to rough COMPLETE ◆ +++ • irregular / --- • Urease () INHIBITION • Gram (+) Rod dendroid margins to spreading • 2-4mm

For in vitro diagnostic use only. This product should be used only by adequately trained personnel with knowledge of microbiological techniques in the laboratory. ©LaMotte BioPaddles. All rights reserved. LaMotte_BioPaddles_R2A_MAC Page | 3 LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

ORGANISM R2A MAC

ORGANISM PHYSIOLOGY GROWTH COLONY IMAGE GROWTH COLONY IMAGE ◆ Precision Test Strip Available Candida albicans • Catalase (+) • White / Opaque • Ascomycete • CVEG / PARTIAL - spreading COMPLETE ------+++ • 4-6mm INHIBITION

E. coli • Lactose (+) • Translucent +++ • Pink / Red • Indole (+) ◆ • CVEG • CVEG • Oxidase (-) ◆ • 0.5 - 1.0mm • 2-4mm ++ • Catalase (+) ◆ • Urease (-) ◆ • Gram (-) Rod) Enterobacter • Lactose (+) • White • Pink aerogenes • Indole (-) ◆ • Thick, round, • Thick, round, • Oxidase (-) ◆ smooth, raised smooth, raised IMAGE • Catalase (+) ◆ to low-convex; to low-convex; PENDING • Urease (-) ◆ +++ Spreading +++ Spreading • Gram (-) Rod • 2-3mm • 2-3mm

Enterococcus spp. • Lactose (-) • Clear • Indole (-) ◆ • CVEG IMAGE • Oxidase (-) ◆ • 1-2mm PENDING PARTIAL - COMPLETE • Catalase (-) ◆ +++ ------INHIBITION • Urease (-) ◆ • Gram (+) Sphere Klebsiella spp. • Lactose (+) • White +++ • Colorless / • Indole (-) ◆ • CVEG Light Pink • Oxidase (-) ◆ • 1-2mm • Spreading +++ IMAGE • Catalase (+) ◆ PENDING • 4-5mm • Urease (+) ◆ • Gram (-) Rod

Proteus spp. • Lactose (-) • White +++ Colorless; • SEE: INDOLE ◆ • CVEG Spreading / • Oxidase (-) ◆ • 1-2mm IMAGE swarming PENDING • Catalase (+) ◆ +++ • 2-3mm • Urease (+) ◆ • Gram (-) Rod

For in vitro diagnostic use only. This product should be used only by adequately trained personnel with knowledge of microbiological techniques in the laboratory. ©LaMotte BioPaddles. All rights reserved. LaMotte_BioPaddles_R2A_MAC Page | 4 LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

ORGANISM R2A MAC

ORGANISM PHYSIOLOGY GROWTH COLONY IMAGE GROWTH COLONY IMAGE ◆ Precision Test Strip Available Pseudomomas • Lactose (-) • Greenish-white • Cream / off- aeruginosa • Indole (-) ◆ and translucent white • Oxidase (+) ◆ • 2-4mm • Irregular

• Catalase (+) ◆ /Spreading IMAGE IMAGE • Urease (-) ◆ +++ +++ • Diffusible PENDING green-blue PENDING • Gram (-) Rod • Fluoresces blue pigment • CVEG under long-wave UV light (400-nm) • 2-4mm

Pseudomomas • Lactose (-) • Clear to amber • Clear or fluorescens • Indole (-) ◆ • CVEG pinkish • Oxidase (+) ◆ • 2-4mm • Irregular • Catalase (+) ◆ edges • Urease (-) ◆ • Clear with • Gram (-) Rod +++ +++ dark, pinkish • Fluoresces blue- centers green under (translucent long-wave UV light edges) (400-nm) • 2-4mm

Salmonella • Lactose (-) • White / +++ • Clear, typhimurium • Indole (-) ◆ Translucent transparent or • Oxidase (-) ◆ • CVEG IMAGE amber IMAGE PENDING PENDING • Catalase (+) ◆ +++ • 2-4mm • Circular, • Urease (-) ◆ Smooth, Flat • Gram (-) Rod • 2-3mm Serratia spp. • Lactose (-) • Pink / Red • Pink / Red • Indole (-) ◆ • CVEG • CVEG • Oxidase (-) ◆ • 2-4mm IMAGE • 2-4mm IMAGE ++ PENDING +++ PENDING • Catalase (+) ◆ • Urease (+) ◆ • Gram (-) Rod

Shigella spp. • Lactose (-) • Colorless or • Indole - mixed ◆ transparent / • Oxidase (-) ◆ PARTIAL - faintly pink IMAGE COMPLETE PENDING • Catalase (+) ◆ ------+++ • Raised INHIBITION • Urease (-) ◆ • 1-3mm • Gram (-) Rod

Staphylococcus • Lactose (-) • Yellowish-gold / aureus • Indole (-) ◆ Opaque PARTIAL - • CVEG COMPLETE • Oxidase (-) ◆ IMAGE +++ • 0.5 - 1.0mm INHIBITION ------• Catalase (+) ◆ PENDING • Urease (-) ◆ • Gram (+) Sphere

For in vitro diagnostic use only. This product should be used only by adequately trained personnel with knowledge of microbiological techniques in the laboratory. ©LaMotte BioPaddles. All rights reserved. LaMotte_BioPaddles_R2A_MAC Page | 5 LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

ENUMERATION

R2A MAC

Very Light Light Moderate Very Light Light Moderate

TVC/TCC TVC/TCC (Total Viable Count/ Total Colony Counts)

Colony Counts <1000 Count colonies TVC/TCC Count = Count x 2.5

Colony Counts >1000 Use chart TVC/TCC Count = Count x 2.5

(Based on a 40 mL sample)

Approximate Colony Count per 100 mL

Example: Inoculated NUT/TTC paddle showing approximately 1000 CFU/100 mL.

For in vitro diagnostic use only. This product should be used only by adequately trained personnel with knowledge of microbiological techniques in the laboratory. ©LaMotte BioPaddles. All rights reserved. LaMotte_BioPaddles_R2A_MAC Page | 6 LaMotte BioPaddles™ TECH DOCUMENT Call: 800-344-3100 Email: [email protected]

DISPOSAL Twist to remove paddle from vial. Fill vial to 40 mL fill line with 1:9 dilution of household bleach (5.25% sodium hypochlorite). Replace paddle in vial. Allow 15 minute contact time. Remove paddle. Discard bleach solution. Replace paddle in vial and dispose. Alternatively, loosen cap and microwave for 30 seconds, autoclave, or incinerate.

GLOSSARY

CVEG Convex, Entire, Glossy FED Full, Entire, Dull Catalase Cat (+) contains catalase enzymes that degrade cellular H2O2. Lactose Lac (+) bacteria can ferment available lactose in the medium producing an acid which lowers the pH. Lac (-) are non-fermenting. Indole Biochemical test to determine the ability of an organism to split indole from the amino acid tryptophan. P. vulgaris is indole (+) while P. mirabilis is indole. Oxidase OX (+) contains cytochrome c oxidase. In contact with an indicator turns dark blue if OX (+); colorless if OX(-). Urease UR (+) presence of enzyme urease which hydrolyzes urea into CO2 & NH4. β-D-Glucoronidase Glu (+) Found in 97% of E. coli strains. The presence of E. coli is determined when both β-D- Glucoronidase and Indole are (+), and the organism is Gram (-). Gram Gram reaction